Evaluation of Polymerase Chain Reaction over Routine Microbial Diagnosis for the Diagnosis of Fungal Keratitis.

SIGNIFICANCE: The significance of the study is that, although conventional culture remains the criterion standard for identifying the causative fungal pathogens, polymerase chain reaction (PCR) may serve as a powerful and high-throughput tool for the early and definitive diagnosis of high-risk patients with mycotic keratitis owing to high sensitivity and specificity.

PURPOSE: This study was focused on comparing the results of PCR with traditional microbial studies for the detection and identification of fungal pathogens in patients with clinically suspected fungal keratitis.

METHODS

Corneal scrapings were collected from 59 patients with clinically suspected fungal keratitis for routine culture, staining, and seminested PCR assay for fungal pathogen identification. The results of PCR were compared with a conventional microbial workup (smear and culture). The samples that were unidentified by culture but were amplified by PCR were further identified by nucleotide sequencing.

RESULTS

Of the 59 patients with suspected fungal keratitis, 38 (64.40%) were found to be positive by PCR assay, 24 (40.67%) by culture, 18 (20.3%) by potassium hydroxide wet mount, and 8 (13.5%) by Gram stains for fungal pathogens. All the 24 isolates found positive with culture were also positive with PCR, so they had not been sequenced for molecular identification. The remaining 14 isolates that were positive with PCR but negative with culture were further identified as Cladosporium cladosporioides, Simplicillium species, Fusarium solani, Alternaria tenuissima, Chaetomium globosum, Penicillium citrinum, and Rhizopus microsporus by sequencing up to the species level.

CONCLUSIONS

The PCR was able to detect the presence of fungal pathogens in a high proportion of culture-negative cases. This study suggests that PCR may serve as a rapid, important complement to traditional culture with high-throughput means of fungal pathogen identification in patients with clinically suspected fungal keratitis.